Rotaviruses are the major cause of life-threatening diarrheal disease in infants and animals worldwide. The long-term research objective of this laboratory is to understand the molecular biology of rotavirus protein function as it relates to mechanisms of pathogenesis and virus assembly. These complex viruses, which lack an envelope, have a unique morphogenetic pathway involving immature particle budding through membranes of the endoplasmic reticulum (ER). Recent studies of the role of nonstructural protein NSP4 in viral morphogenesis led to the discovery that this protein affects calcium homeostasis and functions as an enterotoxin. These studies suggest that NSP4 plays a key role in rotavirus pathogenesis by triggering a cell-signaling pathway that results in diarrhea. A cleavage product of NSP4 that is secreted into the medium of virus-infected cells retains enterotoxin activity. Intracellular NSP4 triggers a distinct signaling pathway that remains to be characterized. Antibody to NSP4 induces broadly cross-protective immunity against rotavirus-induced diarrhea in mice. Thus, NSP4 is clearly an important virulence factor, and virus-induced signaling plays a previously unrecognized role in rotavirus pathogenesis. This grant application proposes studies to understand the molecular details of viral and cellular functions critical for rotavirus pathogenesis. The specific aims of the proposed work are: (1) to dissect the pleiotropic properties of NSP4 by examining the effect of extracellular NSP4 on uninfected epithelial cells; (2) to examine the effects of intracellular NSP4 expression on epithelial cell function; and (3) to understand the role of NSP4 in the morphogenetic process in which virus particles bud through the ER membrane and acquire outer-capsid proteins VP4 and VP7. These studies will provide a molecular foundation to understand rotavirus pathogenesis and viral budding through ER membranes. Understanding these unique aspects of rotavirus pathogenesis offers opportunities to develop new strategies to prevent and control rotavirus disease in children and animals and understand fundamental exocytic processes of eukaryotic cells.